B Cell Translocation Gene 1 Expression Research Articles

The roles of BTG1 mRNA expression in cancers: A bioinformatics analysis.

BTG1 (B-cell translocation gene 1) may inhibit proliferation and cell cycle progression, induce differentiation, apoptosis, and anti-inflammatory activity. The goal of this study was to clarify the clinicopathological and prognostic significances of BTG1 mRNA expression and related signal pathways in cancers. Using the Oncomine, TCGA (the cancer genome atlas), xiantao, UALCAN (The University of ALabama at Birmingham Cancer data analysis Portal), and Kaplan-Meier plotter databases, we undertook a bioinformatics study of BTG1 mRNA expression in cancers. BTG1 expression was lower in gastric, lung, breast and ovarian cancer than normal tissue due to its promoter methylation, which was the opposite to BTG1 expression. BTG1 expression was positively correlated with dedifferentiation and histological grading of gastric cancer (p < 0.05), with squamous subtype and young age of lung cancer (p < 0.05), with infrequent lymph node metastasis, low TNM staging, young age, white race, infiltrative lobular subtype, Her2 negativity, favorable molecular subtyping, and no postmenopause status of breast cancer (p < 0.05), and with elder age, venous invasion, lymphatic invasion, and clinicopathological staging of ovarian cancer (p < 0.05). BTG1 expression was negatively correlated with favorable prognosis of gastric, lung or ovarian cancer patients, but the converse was true for breast cancer (p < 0.05). KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that the top signal pathways included cytokine-cytokine receptor interaction, cell adhesion molecules, chemokine, immune cell receptor and NF (nuclear factor)-κB signal pathways in gastric and breast cancer. The top hub genes mainly contained CD (cluster of differentiation) antigens in gastric cancer, FGF (fibroblast growth factor)-FGFR (FGF receptor) in lung cancer, NADH (nicotinamide adenine dinucleotide): ubiquinone oxidoreductase in breast cancer, and ribosomal proteins in ovarian cancer. BTG1 expression might be employed as a potential marker to indicate carcinogenesis and subsequent progression, even prognosis.

Cystine and Methionine Deficiency Promotes Ferroptosis by Inducing B-Cell Translocation Gene 1.

Ferroptosis is a type of programmed necrosis triggered by iron-dependent lipid peroxidation. We investigated the role of B-cell translocation gene 1 (BTG1) in cystine and methionine deficiency (CST/Met (−))-mediated cell death. CST/Met (−) depleted reduced and oxidized glutathione in hepatocyte-derived cells, increased prostaglandin-endoperoxide synthase 2 expression, and promoted reactive oxygen species accumulation and lipid peroxidation, as well as necrotic cell death. CST/Met (−)-mediated cell death and lipid peroxidation was specifically inhibited by pretreatment with ferroptosis inhibitors. In parallel with cell death, CST/Met (−) blocked global protein translation and increased the expression of genes associated with the integrated stress response. Moreover, CST/Met (−) significantly induced BTG1 expression. Using a BTG1 promoter-harboring reporter gene and siRNA, activating transcription factor 4 (ATF4) was identified as an essential transcription factor for CST/Met (−)-mediated BTG1 induction. Although knockout of BTG1 in human HAP1 cells did not affect the accumulation of reactive oxygen species induced by CST/Met (−), BTG1 knockout significantly decreased the induction of genes associated with the integrated stress response, and reduced lipid peroxidation and cell death in response to CST/Met (−). The results demonstrate that CST/Met (−) induces ferroptosis by activating ATF4-dependent BTG1 induction.

BTG1 inhibits malignancy as a novel prognosis signature in endometrial carcinoma

BackgroundEndometrial carcinoma (EC) is one of the three major malignant tumors of the female reproductive system. In recent years, the incidence and mortality rate of EC have increased. B-cell translocation gene 1 (BTG1) is an anti-proliferation gene that regulates the occurrence and development of a variety of tumors, but there is no research regarding this gene in EC.MethodsBased on The Cancer Genome Atlas (TCGA) database, we used a variety of bioinformatics tools and databases to explore the expression and prognosis of BTG1. We verified expression and prognosis of BTG1 in EC using qRT-PCR and analyzed the relevant clinicopathological parameters. We functionally enriched BTG1 and related genes in EC patients through the bioinformatics website and analyzed miRNA targets of BTG1 and interacting protein networks. Cell proliferation, wound healing, transwell invasion, and cell apoptosis assays were used to detect the effects of BTG1 on the malignant biological behavior of endometrial carcinoma cells (ECCs). The effect of BTG1 on the epithelial-to-mesenchymal transition (EMT) process was detected using western blot.ResultsWe analyzed the expression and prognosis of BTG1 based on TCGA and found that low expression of BTG1 was associated with poor EC prognosis. The qRT-PCR suggested that BTG1 had low expression in EC. BTG1 expression was significantly correlated with overall survival (OS) shortening. Clinicopathological analysis suggested that expression of BTG1 was related to invasion depth and the International Federation of Gynecology and Obstetrics (FIGO) stage. EC pathological tissue type, fertility history, lymphatic metastasis, menopause, estrogen receptor (ER), progesterone receptor (PR), and age of diagnosis were not related. Functional enrichment analysis showed that BTG1 plays an important role in regulating embryonic development, tumorigenesis, apoptosis, and cell cycle. Biological behavior experiments suggest that BTG1 inhibits proliferation, migration, and invasion of ECCs, and promotes apoptosis of ECCs. Western blot indicated that BTG1 inhibited the EMT process of ECCs.ConclusionsBTG1, as a tumor suppressor gene, plays an important role in the occurrence and development of EC. We believe that BTG1 can be used as a potential prognostic biomarker for EC.

HLX affects cell cycle and proliferation in AML cells via the JAK/STAT signaling pathway.

Acute myelogenous leukemia (AML) is a class of malignant tumors derived from hematopoietic stem or progenitor cells. The H2.0-like homeobox gene (HLX) encodes transcription factors that function in promoting normal hematopoietic cell proliferation and tumor immunity. The present study analyzed the effect of downregulating the HLX on cell cycle distribution and cell proliferation in AML. Moreover, the current study detected changes in the expression of genes and proteins in the Janus kinase (JAK)/STAT signaling pathway to investigate the mechanism of the action of HLX in tumor immunity in AML. HLX expression in AML cell lines was silenced using small interfering siRNA, and MTS/PMS-assay colorimetric assays were used to assess the effect of knockdown of HLX on AML cell proliferation. Flow cytometry was used to analyze changes in cell cycle distribution, while reverse transcription-quantitative PCR and western blotting were used to detect changes in the expression levels of key components of the JAK/STAT signaling pathway, such as p21-activated kinase 1 (PAK1), neuropilin 1 (NRP1), B-cell translocation gene 1 (BTG1) and STAT5. It was found that HLX was differentially expressed in AML cell lines of various subtypes, and HLX expression was higher in the AML/M3 subtype NB4 cell line compared with the control group. Knockdown of HLX in NB4 cells significantly inhibited cell proliferation and arrested cells in the G0/G1 phase. Moreover, STAT5 protein expression, as well as NRP1 and PAK1 expression levels were downregulated, while BTG1 expression was upregulated when HLX was knocked out by siRNA. Collectively, the results suggested that downregulation of HLX may cause G0/G1 phase arrest and inhibit the proliferation of AML cells by activating the JAK/STAT signaling pathway.

Prognostic significance of low expression of B-cell translocation gene 1 (BTG 1) in skin squamous cell carcinoma

Although the B-cell translocation gene 1 (BTG1) plays an important role in apoptosis and negatively regulates cell proliferation, BTG1 expression in skin squamous cell carcinoma (SCC) has not been reported. In this study, we wanted to investigate the significance of BTG1 expression in SCC and adjacent tissues. The expression of BTG1 protein and mRNA in SCC tissues and adjacent tissues were detected by immunohistochemistry technique (IHC), Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR). IHC staining showed that the positive expression rate of BTG1 protein in SCC tissues was 54.00%; and the positive rate was 90.50% in the adjacent tissues. Western blot showed that the expression of BTG1 protein in SCC tissues was significantly lower than that in the adjacent tissues (P less than 0.05). RT-PCR showed that the positive rate of BTG1 mRNA in SCC was 50.50%, which was significantly lower than that in adjacent tissues 89.00% (P less than 0.05). Both BTG1 mRNA and protein expression are related to tumor diameter, stage, tumor metastasis and the degree of tumor differentiation in SCC. Patients exhibiting lower BTG1 protein expression in the SCC tissues had a significantly shorter disease-specific survival rate. BTG1 protein expression, tumor diameter, tumors site and stage were independent factors affecting the overall survival of postoperative patients. Further, BTG1 overexpression inhibited A431 cell proliferation ability, while BTG silencing enhanced A431 cell proliferation ability. The lower expression of BTG1 in SCC may be associated with the occurrence, development and prognosis of SCC.

Promoter Methylation Down-regulates B-cell Translocation Gene 1 Expression in Breast Carcinoma

The mechanism responsible for B-cell translocation gene 1 (BTG1) down-regulation in breast carcinoma remains unknown. We examined the BTG1 expression status in breast carcinoma cells and investigated the mechanism underlying the observed alterations. Four breast carcinoma cell lines (SK-BR-3, MDA-MB-231, T-47D, and MCF-7), and one normal mammary epithelial cell line (MCF-10A) were analyzed. BTG1 expression was examined using quantitative reverse transcription polymerase chain reaction (PCR) and western blot. Methylation status of the BTG1 promoter was analyzed using methylation-specific PCR (MSP). To investigate the effect of methylation on BTG1, the cells were treated with a demethylating agent. The carcinoma cells expressed significantly lower levels of BTG1 mRNA and protein than normal cells. The BTG1 promoter was highly methylated in the carcinoma cells. 5-aza-2-deoxycytidine significantly restored BTG1 expression. Down-regulation of BTG1 expression through epigenetic repression is involved in mammary carcinogenesis. BTG1 is a potential diagnostic marker and therapeutic target for breast carcinoma.

Activating transcription factor 4‐dependent B cell translocation gene‐1 induction promotes ferroptosis

Ferroptosis is a special type of programmed cell death that is characterized by iron‐dependent lipid peroxidation. The present study investigated the role of B cell translocation gene‐1 (BTG‐1) on sulfur amino acid deficiency (SAAD)‐mediated cell death. SAAD in HepG2 cells depleted the reduced glutathione, accelerated lipid peroxidation, increased cyclooxygenase 2 expression, and promoted cell death. SAAD‐mediated cell death was specifically inhibited by pretreatment with ferrostain or defroxamine, chemical inhibitors of ferroptosis. In parallel with cell death, SAAD increased expression of microtubule‐associated protein light chain 3‐II and facilitated acridine orange positive autophagic flux. Inhibition of autophagy accelerated the SAAD‐mediated cell death. Moreover, SAAD induced BTG‐1 expression in HepG2 cells. By using BTG‐1 promoter haboring reporter gene and siRNA, activating transcription factor 4 (ATF4) was identified as an essential transcription factor for SAAD‐mediated BTG‐1 induction. Knock out of BTG‐1 gene by CRISPR/Cas9 system decreased the cytotoxicity by SAAD. Furthermore, BTG‐1 deficiency increased microtubule‐associated protein light chain 3‐II and autophagic flux. Thus, present study demonstrates that SAAD induces ferroptosis, and the ATF4‐dependent BTG‐1 induction contributes to accelerate ferroptosis by inhibiting autophagy.Support or Funding InformationThis study was supported by the National Research Foundation of Korea (NRF) funded by Korea government (MSIP)(Grant No. 2018R1A5A2025272 and 2018R1A2B6007514).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The treatment effects and the underlying mechanism of B cell translocation gene 1 on the oncogenesis of brain glioma.

Glioma is characterized by cell over-proliferation, aggressive phenotype, and angiogenesis. B cell translocation gene 1 (BTG1) works as a tumor suppressor in various cancer types. We aimed to study the functions of BTG1 in glioma development. We tested the BTG1 expressions in various glioma cell lines. T98G and U87 cells were transfected with siBTG1 and BTG1 expression vector, respectively, then assessing cell viability, cell migration, and invasion abilities. Flow cytometry was performed to analyze apoptosis and cell cycle distribution. The status of angiogenesis was assessed by In vitro angiogenesis assay. Quantitative polymerase chain reaction and Western blot analysis were used for expression analyses. The inhibitor FH535 (20 μM) and agonist LiCl (20 mM) of the Wnt/β-catenin pathway were introduced to treat transfected cells. BTG1 was low-expressed in glioma cell lines. In T98G cell transfected with siBTG1, cell proliferation, migration, invasion, and angiogenesis abilities were notably increased, BTG1 silencing promoted the cell survival and cell cycle progression from G0/G1 to the S phase. In BTG overexpressed U87 cell, cell proliferation, migration, invasion, and angiogenesis abilities were significantly inhibited. In addition, cell apoptosis significantly increased and the number of G0/G1 phase cells was also significantly increased. We also found that the activation of Wnt/β-catenin was significantly inhibited in BTG1 group. LiCl and FH535 treatments could partially reverse the effects of BTG1 on glioma cell viabilities. Our data suggested that BTG1 functions as a tumor suppressor in glioma, and the anticancer ability of BTG1 involves the repression of the Wnt/β-catenin pathway.

Identification of B-cell translocation gene 1-controlled gene networks in diffuse large B-cell lymphoma: A study based on bioinformatics analysis.

B-cell translocation gene 1 (BTG1) is a member of the BTG/transducer of Erb family. The present study evaluated the impact of BTG1 gene expression on the clinical outcome of diffuse large B-cell lymphoma (DLBCL) and investigated potential mechanisms using the Gene Expression Omnibus (GEO) database. The gene expression profile datasets GSE31312, GSE10846, GSE65420 and GSE87371 were downloaded from the GEO database. BTG1 expression and clinicopathological data were obtained from the GSE31312 dataset. In 498 cases, the expression of BTG1 in DLBCL was associated with treatment response (χ2=19.020; P<0.001) and International Prognostic Index score (χ2=5.320; P=0.025). Using the Kaplan-Meier method, it was identified that the expression of BTG1 was associated with overall survival (OS) and progression-free survival (PFS) times. Univariate and multivariate Cox regression analysis demonstrated that BTG1 was an independent predictive factor for OS and PFS. From the overlapping analysis of 407 BTG1-associated genes and 22,187 DLBCL-associated genes, 401 genes were identified as BTG1-associated DLBCL genes. Pathway analysis revealed that BTG1-associated DLBCL genes were associated with cancer progression and DLBCL signaling pathways. Subsequently, a protein-protein interaction network was constructed of the BTG1-associated genes, which consisted of 235 genes and 601 interactions. Additionally, 24 genes with high degrees in the network were identified as hub genes, which included genes associated with ‘ribosome’ [ribosomal protein (RP) L11, RPL3, RPS29, RPL19, RPL15 and RPL12], ‘cell cycle’ (ubiquitin carboxyl extension protein 52, ATM and Ras homolog family member H), ‘mitogen-activated protein kinase pathway’ (mitogen-activated protein kinase 1), ‘histone modification’ (ASH1-like protein) and ‘transcription/translation’ (eukaryotic translation initiation factor 3 subunit E, eukaryotic translation elongation factor 1 δ, transcription termination factor 1, cAMP responsive element binding protein 1 and RNA polymerase II subunit F). In conclusion, BTG1 may serve as a predictive biomarker for DLBCL prognosis. Additionally, bioinformatics analysis indicated that BTG1 may exhibit key functions in the progression and development of DLBCL.

Cisplatin suppresses proliferation, migration and invasion of nasopharyngeal carcinoma cells in vitro by repressing the Wnt/β-catenin/Endothelin-1 axis via activating B cell translocation gene 1.

Nasopharyngeal carcinoma (NPC) is one of the most commonly diagnosed cancers worldwide with significantly high prevalence in Southern China. Chemoprevention of cancer with alkylating agent compounds could potentially reverse, suppress, or prevent cancer progression. Cisplatin (CIS) is an antineoplastic or cytotoxic platinum-based drug used for chemotherapy of different types of human cancers such as NPC. Nevertheless, the effects of CIS on the migration and invasion of human NPC cells and the underlying molecular mechanisms have not yet been fully scrutinized. In this work, we tested the effect of CIS on the proliferation, migration and invasion of NPC cells. The results exhibited that this drug exerts remarkable inhibitory effects on the proliferation, migration and invasion of NPC cells in a dose-dependent manner. Western blotting and real time RT-PCR were used for expression analyses. We found that CIS treatment led to a dose-dependent inhibition of Endothelin-1 (ET1) expression, at protein as well as mRNA levels in NPC cells. CIS was also found to activate the expression of BTG1 in NPC cells. Moreover, mechanistic analyses revealed that CIS increased the expression of B cell translocation gene 1 (BTG1) to suppress the expression of ET1. Furthermore, we show that ET1 could not be induced in CIS-resistant cells with suppressed BTG1 expression, and subsequently demote the proliferation, migration and invasion of NPC cells. These findings provided compelling evidence of the role of CIS in suppressing NPC metastasis and its underlying molecular mechanisms.

Down-regulation of B-Cell Translocation Gene 1 by Promoter Methylation in Colorectal Carcinoma.

B-cell translocation gene 1 (BTG1) acts as a tumour suppressor in human malignancies. However, the precise mechanism of BTG1 down-regulation in colorectal carcinoma (CRC) remains unclear. We analyzed BTG1 expression in CRC cell lines and tissues and investigated the mechanism underlying the observed alterations. Real-time polymerase chain reaction (PCR) and western blot analyses were performed to analyze BTG1 expression in CRC cell lines. The methylation status of the BTG1 promoter region in cell lines was determined by methylation-specific PCR, and the effect of demethylation on BTG1 expression was explored with 5-aza-deoxycytidine treatment. BTG1 protein expression in CRC tissue samples was evaluated using immunostaining. CRC cell lines and tissue samples expressed lower levels of BTG1 compared to controls, and BTG1 levels were significantly lower in metastatic than primary CRC. In BTG1-down-regulated CRC cell lines, the BTG1 promoter was highly methylated, and 5-aza-deoxycytidine significantly restored BTG1 expression. BTG1 down-regulation in CRC occurs through epigenetic repression, which is involved in the development and progression of CRC.

B-cell translocation gene 1 is downregulated by promoter methylation in ovarian carcinoma.

A better understanding of tumor biology is important in the identification of molecules that are downregulated in malignancy and in determining their role in tumor suppression. B-cell translocation gene 1 (BTG1) has been shown to act as a tumor suppressor in several types of human malignancy. In this study, we analyzed BTG1 expression in ovarian carcinoma cell lines, and we investigated the mechanism underlying the observed alterations. The methylation status of the BTG1 promoter region was determined by methylation-specific polymerase chain reaction, and the effect of demethylation on BTG1 expression was analyzed. BTG1 protein expression in ovarian high-grade serous carcinoma tissue samples was evaluated using immunohistochemistry. BTG1 mRNA and protein expression were reduced in ovarian carcinoma cells. In BTG1-silenced ovarian cancer cells, the BTG1 promoter was highly methylated. Treatment with 5-aza-deoxycytidine significantly elevated BTG1 mRNA and protein expression. Immunostaining demonstrated that BTG1 expression was significantly lower in ovarian carcinoma tissue samples than nonpathological ovaries and fallopian tubes. We demonstrated that BTG1 silencing in ovarian carcinoma occurs through epigenetic repression and is involved in the ovarian carcinogenesis. Our data suggest that BTG1 is a potential therapeutic target for patients with ovarian carcinoma.

Tumor suppressor BTG1 promotes PRMT1-mediated ATF4 function in response to cellular stress.

Cancer cells are frequently exposed to physiological stress conditions such as hypoxia and nutrient limitation. Escape from stress-induced apoptosis is one of the mechanisms used by malignant cells to survive unfavorable conditions. B-cell Translocation Gene 1 (BTG1) is a tumor suppressor that is frequently deleted in acute lymphoblastic leukemia and recurrently mutated in diffuse large B cell lymphoma. Moreover, low BTG1 expression levels have been linked to poor outcome in several solid tumors. How loss of BTG1 function contributes to tumor progression is not well understood. Here, using Btg1 knockout mice, we demonstrate that loss of Btg1 provides a survival advantage to primary mouse embryonic fibroblasts (MEFs) under stress conditions. This pro-survival effect involves regulation of Activating Transcription Factor 4 (ATF4), a key mediator of cellular stress responses. We show that BTG1 interacts with ATF4 and positively modulates its activity by recruiting the protein arginine methyl transferase PRMT1 to methylate ATF4 on arginine residue 239. We further extend these findings to B-cell progenitors, by showing that loss of Btg1 expression enhances stress adaptation of mouse bone marrow-derived B cell progenitors. In conclusion, we have identified the BTG1/PRMT1 complex as a new modifier of ATF4 mediated stress responses.

A novel function of B-cell translocation gene 1 (BTG1) in the regulation of hepatic insulin sensitivity in mice via c-Jun.

Insulin resistance is one of the major factors contributing to metabolic diseases, but the underlying mechanisms are still poorly understood. As an important cofactor, B-cell translocation gene 1 (BTG1) is involved in many physiologic processes; however, the direct effect of BTG1 on insulin sensitivity has not been described. In our study, BTG1 overexpression or knockdown improved or impaired insulin signaling in vitro, respectively. In addition, adenovirus-mediated BTG1 overexpression improved insulin sensitivity in wild-type (WT) and insulin-resistant leptin-receptor mutated (db/db) mice. In addition, transgenic BTG1-overexpressing mice were resistant to high-carbohydrate diet-induced insulin resistance. Adenovirus-mediated BTG1 knockdown consistently impaired insulin sensitivity in WT and insulin-sensitive leucine-deprived mice. Moreover, hepatic BTG1 expression was increased by leucine deprivation via the mammalian target of rapamycin/ribosomal protein S6 kinase 1 pathway. Furthermore, c-Jun expression was up-regulated by BTG1, and adenovirus-mediated c-Jun knockdown blocked BTG1-improved insulin signaling and insulin sensitivity in vitro and in vivo. Finally, BTG1 promoted c-Jun expression via stimulating c-Jun and retinoic acid receptor activities. Taken together, these results identify a novel function for BTG1 in the regulation of hepatic insulin sensitivity and provide important insights into the nutritional regulation of BTG1 expression.- Xiao, F., Deng, J., Yu, J., Guo, Y., Chen, S., Guo, F. A novel function of B-cell translocation gene 1 (BTG1) in the regulation of hepatic insulin sensitivity in mice via c-Jun.

BTG1 potentiates apoptosis and suppresses proliferation in renal cell carcinoma by interacting with PRMT1.

B-cell translocation gene 1 (BTG1) is a member of the BTG/transducer of Erb family. BTG1 regulates cell cycle progression, inhibits proliferation, promotes apoptosis and stimulates cellular differentiation in multiple cell types. However, the functions of BTG1 in renal cell carcinoma (RCC) remain unclear. Therefore, the present study investigated the role of BTG1 in RCC tissue samples and 786-O RCC cells. RCC tissues and cells exhibited significantly weaker BTG1 protein and mRNA expression compared with para-carcinoma control tissues (P<0.05). Upregulated BTG1 expression induced significant G0/G1 cell cycle arrest, apoptosis and inhibition of cell proliferation in 786-O cells (P<0.05). Furthermore, BTG1 interacted with protein arginine N-methyltransferase 1 (PRMT1), and blocking the action of PRMT1 in 786-O cells resulted in inhibition of BTG1 function. These findings indicate that BTG1 may inhibit cell growth and promote apoptosis by interacting with PRMT1 in RCC; the identification of this mechanism may aid in the production of novel therapies for RCC.

B cell translocation gene 1 reduces the biological outcome of kidney cancer through induction of cell proliferation, cell cycle arrest, cell apoptosis and cell metastasis.

The aim of the present study was to determine the expression and function of B cell translocation gene 1 (BTG1) in kidney carcinoma. Kidney samples were obtained from cancer lesions (n=85) and the adjacent normal tissue (n=40) in kidney cancer patients immediately following endoscopic biopsy. The effect of BTG1 overexpression was examined in vitro utilizing a human kidney cancer cell line, ACHN, stably transfected with a recombinant lentivirus (LeBTG1 cells) and compared to empty vector‑transfected controls (LeEmpty). BTG1 protein expression was significantly lower in kidney cancer tissue biopsies compared to normal tissue, as measured by immunohistochemistry (34.1 vs. 77.8% of tissues; P<0.05) and western blotting (0.481±0.051 vs. 0.857±0.081; P<0.05). In vitro analyses revealed that LeBTG1 cells had a reduced survival fraction compared to control LeEmpty cells, with higher rates of apoptosis (16.6±2.5 vs. 6.1±0.7%; P<0.05). The proportion of LeBTG1 cells in G(0)/G(1) stage and S phase was also significantly different from LeEmpty cells (66.8±5.3 and 22.2±1.5% vs. 44.4±3.1 and 34.5±2.3%, respectively; P<0.05), and the migration and invasion of LeBTG1 cells was significantly impaired with respect to LeEmpty cells (74.0±9.0 and 53.0±7.0 vs. 118.0±15.0 and 103.0±13.0, respectively; P<0.05). These effects were accompanied by decreased protein expression of cyclin D1, B‑cell lymphoma 2 and matrix metalloproteinase 9 in LeBTG1 cells (0.118±0.018, 0.169±0.015 and 0.207±0.027, respectively) compared to control LeEmpty cells (0.632±0.061, 0.651±0.063 and 0.443±0.042, respectively; P<0.05). Reduced BTG1 expression is associated with increased disease severity, suggesting it is a negative regulator of kidney cancer and can serve as a prognostic indicator. The results of the present study show that BTG1 protein levels were significantly reduced in kidney cancer biopsy specimens and were associated with disease progression and prognosis.

Diversity of clinical implication of B-cell translocation gene 1 expression by histopathologic and anatomic subtypes of gastric cancer.

Genetic signatures may differ by histopathologic and anatomic subtypes of gastric cancer (GC). B-cell translocation gene 1 (BTG1) was identified as one of genes downregulated in GC tissues from our microarray data. To evaluate the clinical implications of BTG1 expression in GC and the genetic diversity among GC subtypes. BTG1 mRNA expression was analyzed in GC cell lines and 233 pairs of surgical specimens. The mutational and methylation status of BTG1 in GC cell lines was analyzed, and immunohistochemistry was conducted to determine the distribution of BTG1. The pattern and prognostic significance of BTG1 expression were correlated with the three proposed GC subtypes. BTG1 mRNA was downregulated in 82 % of GC cell lines and in 88 % of clinical GC tissues. Promoter hypermethylation events or sequence mutations were not detected in GC cell lines. The pattern of BTG1 expression as observed by immunohistochemistry was consistent with that of its mRNA. Downregulation of BTG1 mRNA in GCs was significantly associated with shorter disease-specific and recurrence-free survival. Multivariate analysis of disease-specific survival identified downregulation of BTG1 transcription as an independent prognostic factor. BTG1 mRNA expression was more strongly suppressed in proximal nondiffuse and diffuse GC compared with distal nondiffuse GC, and subgroup analysis revealed that BTG1 downregulation led to adverse prognosis, specifically in patients with proximal nondiffuse and diffuse GC. Altered expression of BTG1 is a potential biomarker for carcinogenesis and progression of GC, particularly for proximal nondiffuse and diffuse GC.

The Leukemia-Associated Protein BTG1 Is Required for ATF4-Mediated Cellular Stress Responses

During the course of tumorigenesis and subsequent chemotherapeutic intervention, cancer cells experience various kinds of physiological stress, including hypoxia and nutrient limitation. Escaping cell death is one of the routes utilized by these malignant cells to allow continued growth and to acquire therapy resistance. B-cell Translocation Gene 1 (BTG1) is recurrently affected by genomic deletion in pediatric acute lymphoblastic leukemia (ALL) patients. Here, we define BTG1 as a mediator of the cellular stress response. When challenged with cellular stressors, such as amino acid or glucose deprivation as well as drug induced Endoplasmic Reticulum (ER) stress, mouse embryonic fibroblasts (MEFs) lacking Btg1 expression show a 20-30% increased survival rate relative to wildtype cells (Figure 1). Similarly, bone marrow B-cell progenitors isolated from Btg1 knockout mice are more resistant to Asparaginase (ASNase), a drug widely used in the treatment of ALL.Activating Transcription Factor 4 (ATF4) is the master regulator of the stress response pathway that is activated upon nutrient limitation and ER stress. Importantly, loss of ATF4 function results in an enhanced survival almost identical to the effects we measured in Btg1 knockout cells. While ATF4 protein expression itself is not different between the genotypes, gene expression analysis revealed that the induction of a subset of ATF4 target genes (Ddit3, Atf3, Trib3, Gadd34, and Ndrg1) is significantly reduced in Btg1 knockout cells. As these genes are effectors of the apoptosis machinery, increased survival in the Btg1 knockout cells may reflect an attenuation of ATF4 function.We hypothesized that BTG1 complexes with ATF4 to modify its function by recruiting Protein Arginine Methyl Transferase 1 (PRMT1). This enzyme, known to cooperate with BTG1, marks its substrate proteins with a post translational modification but has not been previously implicated in the regulation of ATF4 activity. Co-immunoprecipitation experiments indeed revealed a direct interaction between BTG1 and ATF4. We used purified proteins in an in vitro methylation assay to show that ATF4 is directly methylated by PRMT1 on arginine residue 239. Expression of the mutant ATF4 R239K, which cannot be methylated, in an ATF4 knockout background resulted in reduced transcriptional activity in response to stress relative to wildtype ATF4. In addition, we aimed to mimic the effect of BTG1 loss on the regulation ATF4 function by the addition of PRMT1 inhibitor AMI-1. Treatment of cells with this selective inhibitor faithfully recapitulates BTG1 loss by attenuating the induction of ATF4 target genes upon stress. Our findings establish the interplay of BTG1-ATF4-PRMT1 as a part of the cellular stress response. Taken together, our data indicate that BTG1 is necessary to maintain normal ATF4 function under cellular stress conditions. Loss of BTG1 expression, as it occurs during lymphoid leukemia development, may therefore provide a selective advantage for leukemic cells to survive and to resist treatment at a later stage of disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

B‑cell translocation gene 1 serves as a novel prognostic indicator of hepatocellular carcinoma.

Although the B‑cell translocation gene 1 (BTG1) plays an important role in apoptosis and negatively regulates cell proliferation, BTG1 expression in hepatocellular carcinoma (HCC) has not been evaluated. In this study expression analysis of BTG1 was conducted to clarify the role of BTG1 in the initiation of HCC carcinogenesis and progression. BTG1 mRNA expression levels were determined for HCC cell lines and 151 surgical specimen pairs using quantitative real‑time reverse transcription polymerase chain reaction (RT‑qPCR) assay. The mutational and methylation status of HCC cell lines were analyzed via high resolution melting (HRM) analysis and direct sequencing analysis to elucidate the regulatory mechanisms of BTG1 expression. The expression and distribution of the BTG1 protein in liver tissues were evaluated using immunohistochemistry (IHC). Decreased expression of BTG1 mRNA was confirmed in the majority of HCC cell lines (89%) and clinical HCC tissues (85%) compared with non‑cancerous liver tissues. Mutations or promoter hypermethylation were not identified in HCC cell lines. BTG1 mRNA expression levels were not influenced by background liver status. The pattern of BTG1 protein expression was consistent with that of BTG1 mRNA. Downregulation of BTG1 mRNA in HCC was significantly associated with shorter disease‑specific and recurrence‑free survival rates. Multivariate analysis of disease‑specific survival rates identified BTG1 mRNA downregulation as an independent prognostic factor for HCC (hazard ratio 2.12, 95% confidence interval 1.12‑4.04, P=0.022). Our results indicate that altered BTG1 expression might affect hepatocarcinogenesis and may represent a novel biomarker for HCC carcinogenesis and progression.

735P - B-Cell Translocation Gene 1 Serves As a Novel Prognostic Indicator of Hepatocellular Carcinoma

ABSTRACT Aim: Although B-cell translocation gene 1 (BTG1) plays an important role in apoptosis and negatively regulates cell proliferation, BTG1 expression in hepatocellular carcinoma (HCC) has not been evaluated. The aim of this study was to clarify the role of BTG1 in the initiation of HCC carcinogenesis and progression. Methods: BTG1 mRNA expression levels were determined for HCC cell lines and 151 surgical specimen pairs using a quantitative real-time reverse transcription polymerase chain reaction assay. The mutational and methylation statuses of HCC cell lines were analyzed via high-resolution melting analysis and direct sequencing analysis to explore the regulatory mechanisms of BTG1 expression. The expression and distribution of the BTG1 protein in liver tissues were evaluated using immunohistochemistry. Results: Decreased expression of BTG1 mRNA was confirmed in the majority of HCC cell lines (89%) and clinical HCC tissues (85%) compared with non-cancerous liver tissues. Mutations or promoter hypermethylation of BTG1 were not identified in HCC cell lines. BTG1 mRNA expression levels were not influenced by background liver status. The pattern of BTG1 protein expression was consistent with that of BTG1 mRNA. Downregulation of BTG1 mRNA in HCC was significantly associated with shorter disease-specific and recurrence-free survival rates. Multivariate analysis of disease-specific survival rates identified BTG1 mRNA downregulation as an independent prognostic factor for HCC (hazard ratio 2.12, 95% confidence interval 1.12 – 4.04, P = 0.022). Conclusions: Our results indicate that altered BTG1 expression might affect hepatocarcinogenesis and may represent a novel biomarker for HCC carcinogenesis and progression. Disclosure: All authors have declared no conflicts of interest.